The present invention relates to systems for the magnetic collection of magnetically-responsive particles (referred to herein as “magnetic particles”), and the subsequent controlled dispersion of the particles. In particular, the invention relates to the detection, isolation, separation and/or manipulation of target substances, such as for example, chemicals and/or biological substances such as biochemicals, cells, cell components, bacteria, viruses, toxins, nucleic acids, hormones, proteins, receptor-ligand complexes, other complex molecules, or combinations thereof, by selective interaction thereof with magnetic particles, which can be separated from a medium, and subsequently resuspended for further analysis, isolation or other use.
As a background to the invention, many techniques are known and used in the prior art that involve identification, separation, and/or manipulation of target entities, such as cells or microbes, within a fluid medium such as bodily fluids, culture fluids or samples from the environment. It is readily appreciated that such techniques often require multiple washing and binding steps. These steps are conducted in many such techniques by tedious manual pipetting and decanting procedures or by using automated robotic systems that are large and not field-portable. Such identification, separation and/or manipulation techniques may involve, for example, analyzing a sample to determine whether a specific pollutant, toxin or other substance is present and, if so, in what quantity; extracting a specific target substance, such as nucleic acid fragments, proteins, enzymes or the like from a sample for subsequent processing or utilization; or the like. It is readily appreciated that such techniques often must exhibit significant sensitivity, especially when the target substance is present in trace quantities, in order to provide accurate and useful information or to provide a suitable specimen in an isolation technique.
One example of a technique that involves separating, isolating or otherwise manipulating organic molecules in trace is an immunoassay technique using antibodies as the analytical reagents. The principle of immunoassays is well understood. Antibodies recognize a specific analyte by following specific interactions. For low molecular weight analytes such as drugs or metabolites, it is customary to perform competitive immunoassays. Typically, a fixed, limited quantity of specific antibody is allowed to incubate with a known concentration of labeled analyte and a sample possibly containing some unknown concentration of that analyte. The quantity of label bound to antibody is inversely proportional to the amount of analyte in the test specimen.
Although there is a high number of immunoassay techniques, only a few are widely used. Among these are indirect techniques where the analyte is measured through a label species conjugated with one of the immunoreagents. For quantitation, it is customary to perform a bound/free separation so that labeled analyte associated with the antibody can be detected. If one of the components of the immunologic reaction is immobilized on a solid phase (heterogeneous assay), the experimental procedure is simplified. A heterogeneous immunoassay that involves competitive binding of the analyte and an enzyme-labeled analyte to the antibody is called an Enzyme Linked Immunosorbent Assay (ELISA).
For analytes which have at least two distinguishable antigenic determinants, a simpler and more precise approach is to perform a sandwich immunoassay, which uses a first antibody directed to one antigenic site as a capture antibody and a second antibody directed at another characteristic determinant as the signal generating antibody. Thus, if the capture antibody is separated from solution, or bound on some solid support, the only way in which signal antibody can be bound to solid support or separated from solution is via analyte. The advantages of sandwich assay are that: (1) signal is directly proportional to analyte concentration on the low end of the analyte curve; (2) extreme sensitivity can be obtained on the low concentration end; (3) sandwich assays are assays of “excesses” since capture antibody and label antibody are typically in excess of analyte and so error is mainly related to accuracy of sample input; and (4) a wide dynamic analyte detection range (as much as 4-5 logs) is possible. Sandwich assay technology, like competitive assay, employ a wide range of systems for performing bound/free separations. Typically in such assays, antibodies for the analytes of interest are placed with great precision on a solid support so as to permit analyte binding to take place on the bound antibodies. Next, solution is added which causes unbound and non-specifically bound analyte to be carried from the binding region. Then a second labeled antibody is added, and solution is added to wash away the unbound secondary antibody. If the secondary antibody is enzyme labeled, then enzyme substrates are added to result in a detectable signal (e.g. color change, fluorescence, conductivity change) which will be proportional to the quantity of enzyme (and therefore analyte) specifically bound.
There are numerous ways for performing the bound/free separation utilizing a specific binding substance immobilized on a solid phase, such as, for example, antibody adsorbed or covalently linked to the inside of a tube (coated tube assay), or, more recently, affixed to a mobile solid phase, such as, for example, elongate structures that can be submerged in a liquid and then withdrawn, or beads, which can either be centrifuged or separated with filters or magnetically. Typically, a separation system should have the characteristics that the separation can easily be performed, excess reagent rates for DNA hybridization and elution, as well as for DNA amplification using polymerase chain reaction (PCR) or other enzyme amplification methe.
Detection of signal or radiant-energy response in an assay such as those discussed above may be accomplished through a variety of techniques. One example is fluorescent detection of a fluorescently labeled antibody, analyte or other small molecule that could be associated with the analyte. Radioactive detection is also a possibility if system components are impervious to the type of radioactive emission detected. Colorimetric detection of a dye attached to the antibody or analyte, possibly enclosed in a liposome, is also possible. Chemiluminescent, bioluminescent, electrochemiluminescent, or enzymatic detection is also possible, provided the substrate for the detection reaction become available after the bound/free separation. Thus, a variety of methods exist to obtain a readable signal or other radiant-energy response.
One major challenge in the use of immobilized binding substances is the limited lifetime of a chemically selective surface, especially those that include biomolecules (often referred to as “biosensors”). Many biomolecules in aqueous solution at room temperature will chemically degrade over time and typically have a lifetime ranging from only hours up to about 1 week. Therefore, it is not feasible to keep a biosensor surface immersed in aqueous solution continuously for long periods of use. The solution composition also effects the lifetime of biosensors. The structure and function of biomolecules and biological materials are sensitive to environmental conditions such as salt concentration, pH and temperature. Changes in solution composition and temperature can irreversibly denature proteins so that they will no longer bind to specific ligands. The use of whole cells in sensor systems is also challenging since cells require the correct mix of metabolites to remain viable, and solution composition and solution flow rate effect the growth rate of viable cells. The specificity and therefore irreversible nature of many biospecific interactions also contributes to the limited lifetime of biosensing surfaces. Selective interactions such as antibody-antigen interactions are essentially irreversible over the time course of minutes. Harsh reagents may be used to remove bound antigens (and non-specifically bound molecules); however, this decreases the subsequent binding activity of the antibody itself so that the regenerated sensing surface is not as effective as the fresh sensing surface, negatively impacting assay accuracy and reliability. In addition, the lifetime of a biosensor is often also limited due to “fouling” of the sensor surface caused by the non-specific binding of materials in a sample matrix onto the biosensor surface.
These problems have resulted in increased use of renewable surfaces for biosensing in which the chemically selective chemistry is on the surface of small particles. Fresh aliquots of derivatized particles can therefore be used for each analysis. After such an analysis, the derivatized particles can be flushed from the system and new particles are used for a subsequent analysis.
Where the derivatized particles are magnetic particles, isolation of the particles from media during analysis can be achieved using magnetic separation or high gradient magnetic separation (HGMS). As used herein, the term “magnetic” is intended to refer to a property of a particle whereby a force is exerted thereon by the application of a magnetic field thereto. In magnetic separation, particles of relatively larger size (i.e., about 0.5 microns or greater in diameter) are captured or separated and in HGMS, smaller particles, such as, for example, colloidal magnetic particles are separated.
Over the past several years, sub-millimeter-scale, automated flow-based analyzers and chemical detector arrays have steadily approached the technology level needed for commercialization. Development is continuing toward ever more compact diagnostic analyzers for automated immunoassays, DNA purification and amplification, cell separation, environmental contaminant detection and the like.
In light of this background, there remain needs for further development of systems for the magnetic collection of magnetic particles, and the subsequent controlled resuspension of the particles. In particular, a need exists for further development of systems for the specific detection, isolation, separation and/or manipulation of target substances by selective interaction thereof with magnetic particles, which can be separated from a medium, and subsequently resuspended for further analysis, isolation or other use. The present invention addresses these needs.